Solid-phase synthesis and pathological evaluation of pyroglutamate amyloid-β3-42 peptide

Pyroglutamate amyloid-β3-42 (AβpE3-42) is an N-terminally truncated and pyroglutamate-modified Aβ peptide retaining highly hydrophobic, amyloidogenic, and neurotoxic properties. In Alzheimer’s disease (AD) patients, AβpE3-42 peptides accumulate into oligomers and induce cellular toxicity and synaptic dysfunction. AβpE3-42 aggregates further seed the formation of amyloid plaques, which are the pathological hallmarks of AD. Given that AβpE3-42 peptides play critical roles in the development of neurodegeneration, a reliable and reproducible synthetic access to these peptides may support pathological and medicinal studies of AD. Here, we synthesized AβpE3-42 peptides through the microwave-assisted solid-phase peptide synthesis (SPPS). Utilizing thioflavin T fluorescence assay and dot blotting analysis with anti-amyloid oligomer antibody, the amyloidogenic activity of synthesized AβpE3-42 peptides was confirmed. We further observed the cytotoxicity of AβpE3-42 aggregates in cell viability test. To examine the cognitive deficits induced by synthetic AβpE3-42 peptides, AβpE3-42 oligomers were intracerebroventricularly injected into imprinting control region mice and Y-maze and Morris water maze tests were performed. We found that AβpE3-42 aggregates altered the expression level of postsynaptic density protein 95 in cortical lysates. Collectively, we produced AβpE3-42 peptides in the microwave-assisted SPPS and evaluated the amyloidogenic and pathological function of the synthesized peptides.

Here, we introduce a facile synthetic method of Aβ pE3-42 production utilizing microwave-assisted solid-phase peptide synthesis (SPPS) 15 . To test aggregation propensity of the synthesized Aβ pE3-42 peptides, we used in vitro assays, thioflavin T (ThT) assay and dot blotting analysis with anti-amyloid oligomer antibody. We tested the cellular toxicity of the synthesized Aβ pE3-42 peptides in the cell viability assay. We then intracerebroventricularly injected Aβ pE3-42 oligomers into imprinting control region (ICR) mouse models and performed behavior tests, Morris water maze and Y-maze tests, to identify cognitive deficits induced by synthesized Aβ pE3-42 peptides. We further obtained brain lysates from the Aβ pE3-42 -infused mouse models and analyzed the alteration of the synaptic protein marker levels by the western blot method.

Results and discussion
Synthesis of Aβ pE3-42 peptides. Although Aβ pE3-42 peptide is emerging as an attractive target for AD research, an effective synthetic method of this peptide is not yet developed. For the facile and reproducible production of Aβ pE3-42 , we utilized previously-reported Aβ synthesis protocol using a microwave synthesizer for the preparation of Aβ pE3-42 peptide 16 . Microwave-assisted SPPS improves the efficiency of the peptide synthesis due to reduce the reaction time and to suppresses the intermolecular aggregation, resulting in the inaccessibility of the coupling reagents to react with the peptide 17,18 . Compared to the general approach of SPPS, microwave heating method increases the peptide yield from 20 to 70% and reduces the amino acid coupling time to 5 min (min) 15 . In microwave-assisted SPPS, we synthesized Aβ pE3-42 peptide in 6.5 h (h) and obtained 71% peptide yield. This result showed that our synthetic approach minimizes the reaction time and enhances the peptide yield of Aβ pE3-42 . To conjugate the first C-terminal amino acids to the resin, we used the symmetric anhydride activation, and the following amino acids were sequentially synthesized to produce N-terminally truncated Aβ 4-42 peptides and additionally coupled pyroglutamates (Fig. 1). This approach brings in benefits to avoid complicated N-terminal cleavage and glutamate cyclization steps 14 . We further purified Aβ pE3-42 peptides adapting reverse phase-high performance liquid chromatography analysis ( Supplementary Fig. S1).
Aggregation properties of synthetic Aβ pE3-42 . During the progression of AD, Aβ monomers aggregate into soluble oligomers and further become insoluble fibrils, the main components of the cerebral plaques in the brains 19 . As the misfolding and assembly is the key pathological feature of Aβ in AD, it is critical to evaluate the aggregation properties of synthetically obtained peptides. Thus, to identify the aggregation propensity of the synthesized Aβ pE3-42 peptide, we performed ThT fluorescence assay. ThT intercalates into the β-sheet formation of Aβ fibrils, and the shifted fluorescence intensity of ThT upon β-sheet was measured to analyze the amounts of Aβ fibrils 20 . We prepared 25 μM of Aβ pE3-42 samples in deionized water with 5.5% dimethyl sulfoxide (DMSO) and incubated during the time range from 0 to 96 h at 37 • C. ThT solution (5 μM) was added to each of incubated Aβ solution samples and measured the intensity of ThT (Fig. 2a). We observed a saturation phase at 19 h of incubation, indicating the formation of β-sheet-rich Aβ pE3-42 fibrils. We further compared the aggregation propensity of the synthesized Aβ pE3-42 peptide with that of Aβ 1-42 and Aβ 4-42 peptides, which rapidly form aggregates 21 . Both of Aβ 1-42 and Aβ 4-42 peptides were produced through the previously reported synthesis protocol ( Supplementary  Fig. S2) 16 . In ThT assay, Aβ pE3-42 formed β-sheet rich fibril after 19 h of incubation time, whereas Aβ 1-42 and Aβ 4-42 aggregated into fibrils after 23 and 28 h of incubation, respectively ( Supplementary Fig. S3). This result was consistent with the previous report that N-terminal deletion or modification of Aβ 1-42 enhances the aggregation of N-terminal truncated Aβ peptide compared to the Aβ 1-42 21 . As ThT assay is limited to detect β-sheet-rich fibrils, www.nature.com/scientificreports/ we performed dot blotting analysis using anti-amyloid oligomer A11 antibody for monitoring the formation of synthesized Aβ pE3-42 oligomers 22 . We prepared 2 μL of identical Aβ pE3-42 samples used in ThT assay and loaded on the nitrocellulose membrane and performed dot blotting analysis (Fig. 2b). The levels of Aβ pE3-42 oligomers were gradually increased until 96 h incubation. Interestingly, we observed oligomers even before the incubation at 37 • C (0 h incubation sample). This result supports the previous study that monomeric Aβ pE3-42 sample has aggregates due to the spontaneous aggregation process of the peptide 23 . Overall, we examined the amyloidogenic properties of the synthesized Aβ pE3-42 peptides in both of ThT assay and dot blotting analysis. We quantified the level of cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay ( Fig. 3) 24 . Cell viability of Aβ 1-42 , Aβ 4-42 , and Aβ pE3-42 groups were statistically compared to nontreated group (n = 3 per group). For the statistical analysis, we performed one-way ANOVA analysis followed by  www.nature.com/scientificreports/ Bonferroni's post-hoc comparison tests. Compared to the non-treated group, the cell viability was decreased to 48.1% (p < 0.0001) in Aβ pE3-42 -treated group, 49.9% (p < 0.0001) in Aβ 1-42 -treated group, and 62.3% (p < 0.0001) in Aβ 4-42 -treated group. This result showed that all the Aβ variants initiates cellular toxicity in the aggregated form. In cell viability assay, we confirmed that the synthetic Aβ pE3-42 aggregates induce severe cellular toxicity in mouse hippocampal neuronal cells.

Cognitive impairments induced by synthetic Aβ pE3-42 in rodents.
To confirm the neuropathological function of the synthesized Aβ pE3-42 peptide, we injected Aβ pE3-42 oligomers into the lateral ventricular region of ICR mice as injected soluble Aβ oligomers inducing neurotoxicity and cognitive decline 25 . For comparison, we used Aβ 1-42 peptide, one of the major pathological forms of Aβ variants associated with cognitive decline 26 , as a control. Aβ 1-42 peptide was synthesized and purified through the previously reported protocol 16 . Since the dot blotting analysis result showed that soluble oligomers were clearly appeared after 8 h of incubation, we incubated 25 μM of Aβ solution for 8 h at 37 • C to prepare oligomeric samples for the injection. We conducted 5 μL intracerebroventricular (ICV) injections of blank saline, Aβ pE3-42 oligomer solution (10 μM), and Aβ 1-42 oligomer solution (10 μM) into brains of seven-week-old male ICR mice. After five days of recovery time, we performed learning and memory behavior tests, Y-maze and Morris water maze tests (Fig. 4a). All results from behavior tests were statistically compared to vehicle group (n = 7 per group). For the statistical analysis of the behavior tests, one-way ANOVA analysis followed by Bonferroni's post-hoc comparison tests were performed. Y-maze is widely applied to measure the short-term memory function of mouse models due to they normally explore new arm entries of the maze rather than the arm that was previously entered 27 . We found that both of Aβ pE3-42 (p < 0.0001) and Aβ 1-42 (p < 0.001) oligomers induced short-term memory impairments compared to , and (k) total swimming distance of all groups. All data were statistically compared to vehicle group. One-way ANOVA analysis followed by Bonferroni's post-hoc comparison tests were performed in all statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 www.nature.com/scientificreports/ vehicle (saline-injected) groups as the alternation levels were decreased (Fig. 4b). Total arm entry data showed that there were no significant differences of arm entries among all groups (Fig. 4c). After the Y-maze test, we performed Morris water maze test to examine the spatial learning dysfunction by Aβ pE3-42 oligomers 28 . During the five days of practice, we placed mice on four different quadrants in the water tank and measured time to reach the hidden platform for surviving in the water tank. In the trial day, we removed the platform and measured the crossing number, time in platform location, and time in zone. Compared to Aβ pE3-42 and Aβ 1-42 groups, the escape latency of the vehicle groups was reduced on the fourth day of training (Fig. 4d). On the probe trial day, platform crossing number and time in platform of Aβ pE3-42 (p < 0.01) and Aβ 1-42 groups (p < 0.01) were decreased comparing to the vehicle groups (Fig. 4e-i). We also analyzed the time in target quadrant and found that saline groups stay in the target quadrant longer than Aβ pE3-42 (p < 0.05) and Aβ 1-42 groups (p < 0.05) (Fig. 4j). There were no significant differences of swimming distance among all groups (Fig. 4k). Overall, results from both of Y-maze and Morris water maze tests showed that the synthesized Aβ pE3-42 aggregates induced impaired short-term and spatial memory function of mice.
Synaptic protein alteration by synthetic Aβ pE3-42 . As the behavior tests showed that synthesized Aβ pE3-42 disrupted cognitive function of ICR mouse models, we sacrificed the ICV-injected mice and prepared brain lysates to further observe the changes of synaptic protein levels by Aβ pE3-42 aggregates. Postsynaptic density protein 95 (PSD-95) regulates the synaptic strength of post-synaptic membrane, and synaptophysin supports synaptic vesicle endocytosis 29,30 . Both of proteins are highly related to the synaptic plasticity and widely used biomarkers for cognitive function. In order to examine such alterations in synaptic plasticity, we used a western blot method to analyze PSD-95 and synaptophysin expression levels in the brain lysates of Aβ pE3-42 , Aβ 1-42 , and saline groups (Fig. 5a). All the quantification of blots were statistically compared to vehicle group (n = 4 per group). For the statistical analysis of the quantification, one-way ANOVA analysis followed by Bonferroni's post-hoc comparison tests were performed. PSD-95 expression levels were decreased within the cortex region of Aβ pE3-42 (p < 0.0001) and Aβ 1-42 groups (p < 0.001) compared to the saline groups (Fig. 5b). However, there was no change of synaptophysin levels among three groups (Fig. 5c). In the hippocampus region, both PSD-95 and synaptophysin expression levels were not significantly altered in Aβ pE3-42 and Aβ 1-42 groups (Fig. 5d,e). All results from the western blot assay showed that the synthesized Aβ 1-42 and Aβ pE3-42 oligomers suppress postsynaptic memory function in cortical region of the mice.

Conclusion
In this study, we investigated a facile synthetic method of Aβ pE3-42 peptides and confirmed the amyloidogenic and pathologic properties of the synthesized peptides. In ThT assay and dot blotting analysis, we found that Aβ pE3-42 peptides rapidly accumulate into oligomers and fibrils. We further examined cytotoxicity of synthetic Aβ pE3-42 aggregates. Utilizing Aβ pE3-42 -infused mouse models, we performed Y-maze and Morris water maze tests and observed synaptic dysfunction induced by synthetic Aβ pE3-42 oligomers. Furthermore, the expression level of PSD-95 was decreased in the cortical region of Aβ pE3-42 -infusion mouse models compared to the vehicle groups. As Aβ pE3-42 aggregates are highly associated with AD, the presented synthetic method of Aβ pE3-42 will support pathological, therapeutic, and diagnostic investigation of the disorder. Safety statement. TFA is an acid, which severely irritates and burns the skin and eyes. Furthermore, breathing TFA will damage nose and throat. Thus, the protective clothes, googles, and gloves are required before treating TFA.

Methods
Synthesis of Aβ pE3-42 peptides. Aβ 4-42 peptide was synthesized through the previous Fmoc SPPS protocol 16 . For the first C-terminal alanine coupling, symmetric anhydride activation was utilized. We ThT fluorescence assay. To monitor the aggregation propensity and quantify the β-sheet formation in Aβ pE3-42 aggregates, ThT fluorescence assay was performed. Aβ pE3-42 peptide was dissolved in DMSO (1 mM) and diluted with deionized water to make Aβ pE3-42 solution (25 μM After removing the primary antibody and washing the membrane with TBST for three times and added HRP-conjugated goat anti-rabbit secondary antibody (1:10,000, Bethyl Laboratories, USA) and incubated for 1 h at room temperature. Membranes were washed three times with TBST, and the proteins on the membranes were developed using ECL kit (Thermo Fisher Scientific, USA).
Cell viability assays. HT

Y-maze.
For observing the short-term memory dysfunction initiated by Aβ pE3-42 oligomers, Y-maze test was performed. The maze was built with three arm entries (40 cm long, 10 cm wide, 12 cm high), which were symmetrically disposed at 120° angles. Each of mouse was placed at the edge of the arm entry and allowed to move around the maze for 8 min. After recording the experiment, the number of total arm entries and sequence of arm choices were analyzed. The percent alternation was calculated by the proportion of arm choices that differ from the previous two choices. After the trial, the maze was cleaned with 70% ethanol solution before the next trials.
Morris water maze. Morris water maze tests was performed to monitor deficits of spatial learning function induced by Aβ pE3-42 oligomers. The maze contained a circular water tank (120 cm diameter, 25 ± 1 • C) with the hidden quadrant, which was placed 1.5 cm below the water surface. The maze is equally divided into four quadrants (A,B,C,D) and three different shapes of cues are located on the top of quadrant B, C, and D to provide the direction for the mice. The hidden platform is placed on the middle of quadrant C and mice can survive if they reach the platform. The tank is filled with water with non-toxic white paint to prevent the chances that mice can see the hidden platform. We performed five days of training with four trials per day. In each trial, mouse was placed at the end of quadrant and allowed to swim and reach the platform for 60 s . After 60 s of trial, we took the mouse out of the maze and provided 10 min of resting time before the next trial. We measured escape latency, which is the average of the time to reach the platform during four trials. On the sixth day, we performed 60 s of probe test. Each mouse was placed at the edge of the opposite quadrant of the target zone. The hidden platform was removed and we analyzed crossing number, time in platform, time in the target quadrant, and swimming distance.
Lysate preparation. After the behavior tests, we sacrificed all the mice to prepare brain lysates. We dissected the hippocampal and cortical regions of brains. All the brain lysates were homogenized in ice-cold RIPA buffer with 1X protease inhibitor cocktail. Homogenized brain lysates were incubated in ice for 20 min and collected the supernatants after the centrifugation at 14 000 rpm at • C for 30 min. The protein concentrations in supernatants were quantified via Pierce BCA protein assay kit.

Western blot.
To analyze the alternation of synaptic protein marker levels, PSD-95 and synaptophysin levels, by Aβ pE3-42 aggregates, we conducted the western blot analysis. For the experiment, 15 μg of cortical and hippocampal brain lysates were loaded on the 12% gel and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We transferred the proteins from the gel to a nitrocellulose membrane and the membranes were blocked with a 5% skim milk solution for 1 h. After the blocking, we washed the membrane with TBST (tris-buffered saline with 0.1% Tween 20) for three times and incubated with primary antibody PSD-95 (1:2000, Invitrogen, USA) or synaptophysin (1:1000, MilliporeSigma, USA) for overnight at 4 • C. After removing the primary antibody and washing the membrane with TBST for three times and added HRP-conjugated goat antimouse secondary antibody (1:10000, Bethyl Laboratories, USA) and incubated for 2 h at room temperature. Membranes were washed three times with TBST, and the proteins on the membranes were developed using ECL kit (Thermo Fisher Scientific, USA). For control of the protein loading levels, we used β-actin (1:2000, Mil-liporeSigma, USA).